Proteomics and Mass Spectrometric Analysis Laboratory

Proteomics and mass spectrometric analysis of biologics

At the Centre for Vaccine Evaluation (CVE), we have developed scientific expertise in various techniques that help provide new tools for evaluating biologics. We use mass spectrometry and proteomics as key approaches to studying proteins.

 

Why we study protein samples and mixtures

For biologics, such as vaccines and blood products, proteins are the critical components. Proteins are the biologically active ingredients that allow the product to work, just as acetylsalicylic acid (ASA) is the active ingredient in Aspirin®. To evaluate the quality of a biologic product (for example, to correctly identify quantities of active ingredients as well as impurities), samples containing small sets of compounds as well as complex biologics, such as vaccines, must be characterized. Large sets of proteins present in the blood or tissue of the recipient of a biotherapeutic must also be studied to provide insight into determinants of safety and efficacy for the biologic. For example abnormal levels of a set of blood proteins that are responsible for a specific function may be reflective of a disease state or adverse reaction.

 

How we study protein samples in the proteomics and mass spectrometric analysis laboratory

At the Centre for Vaccine Evaluation (CVE), the scientific expertise in the Proteomics and Mass Spectrometric Analysis Laboratory is used to tackle questions that involve protein analysis. One key approach involves developing and applying mass spectrometry (MS) methods to analyse biologics. The laboratory's research and analytical expertise is used to identify thousands of proteins present in a sample or expressed in a given tissue or cell type. It is also possible to provide a detailed characterization of unique proteins or small sets of proteins. The combined approach of large-scale and small-scale analyses represents a great asset. For instance, when facing a timely issue involving thousands of proteins, a proteomics approach provides a valuable overview of what is going on at the protein level as well as direction in resolving biological questions. In the case of purified proteins, a detailed peptide sequence provides valuable insights when more information is needed.

CVE's work in this area helps in:

  • Identifying and characterizing ingredients in biologics to provide information on the quality, safety and efficacy of a product;
  • Developing and improving techniques for studying subsequent entry biologics (SEBs);
  • Providing rapid analysis of urgent samples such as those related to severe, unexpected adverse reactions;
  • Correlating diseases with changes in a proteome;
  • Developing and improving techniques for vaccine testing.

Most of the MS-based projects deal with developing and applying high-performance liquid chromatography (HPLC) separation methods directly coupled to high-accuracy tandem mass spectrometers (MS/MS spectrometers) to identify and characterize complex mixtures of organic compounds. The types of analytes range from intact glycoproteins, peptide maps (enzymatic digests of a protein into a characteristic group of peptides) and proteome analysis (simultaneous analysis of multiple peptide maps) to mixtures of unknown counterfeit drugs.

Currently, research projects in CVE that use mass spectrometry fall under two main categories:

  • Proteomics projects that identify and measure large numbers of proteins present in complex samples;
  • Recombinant protein characterizations that include characterization of peptide sequences, disulphide bonds, and post-translational modifications, such as sulphonation, phosphorylation and glycosylation.

 

Concepts and tools we use to study proteomics and mass spectrometric analysis of biologics

At the Centre for Vaccine Evaluation (CVE), we use mass spectrometry (MS) as an extremely sensitive analytical technique that separates and accurately measures the mass-to-charge (m/z) ratio of ions.

Two recent dramatic innovations in this field have helped apply this method to biologics:

  • The advent of new ionization methods, such as electrospray ionization (ESI), has allowed the generation of gas phase protein ions in the mass range of the instrument;
  • Technological developments for the separation of gas phase ions, such as time-of-flight (TOF) mass analysers, have allowed for rapid and highly accurate mass measurement of proteins and peptides.

TOF analysis is performed by accelerating a set of ions from the inlet area of the mass spectrometer toward the detector. Basic physics principles dictate that the lighter fragments (that is, those with the lower mass) arrive at the detector first. Therefore, the time from acceleration to detection for any population of ions can be translated into a calculated m/z ratio.

The combination of ESI and TOF allows for determination of the mass of the ionized or charged particles. However, since many different molecules can have a similar weight, mass determination alone is rarely enough information for confident identification of a compound. That is why an extra parameter, ion fragmentation, is analysed.

The general process for MS analysis involves ionizing a sample in an ion source to generate charged, gas phase ions from the molecules in the sample. The ions are separated by m/z in a mass analyser. To examine ion fragmentation, a tandem MS (MS/MS) approach is used. In this approach, populations of ions with a particular m/z ratio are isolated and then fragmented by gas phase collisions. The fragments from the selected ion population are then analysed in the mass spectrometer. MS/MS is commonly applied to the analysis of peptides. It lets one determine amino acid sequence information, which is much more useful for protein identification than mass determination.

Mass spectrometry has become a powerful technique in the study of proteins largely because of its tremendous sensitivity. Current instruments are capable of identifying proteins at concentrations as low as 10 femtomoles (0.000 000 000 2 grams for a protein with a molecular weight of 20 000 grams/mol).

Some applications of MS include:

  • Identifying proteins;
  • Detecting contaminants in samples;
  • Protein sequencing (that is, identifying and determining the order of the amino acid building blocks that make up proteins);
  • Identifying changes in a protein that occurred after it was translated from the genome (post-translational modifications) such as phosphorylation and glycosylation.

Proteomics refers to the study of sets of proteins expressed in a specific cell, tissue or organism. These complete sets of proteins are also known collectively as a proteome. Many factors can affect the type, structure and level of proteins that are expressed and present. Changes in a proteome can have varied effects.

The study of the proteome includes:

  • Separating and identifying proteins;
  • Determining the relative concentration of proteins;
  • Examining protein composition;
  • Determining protein structure;
  • Investigating how proteins interact with each other.

The methodology may be applied to naturally occurring components such as blood as well as therapeutic products such as biologics, which can contain thousands of proteins. For example, blood plasma contains thousands of different proteins that range by a factor of 1012 in concentration; whereas the proteome of a monovalent vaccine contains a much smaller number and concentration range of proteins from a specific organism that is being immunized against.

 

Research highlight 1: Detection of contaminants - rapid response to adverse reactions and impurities

At the Centre for Vaccine Evaluation (CVE), separation science is used to provide a rapid and detailed analysis of the compounds in a biologic sample. Separation science helps to confirm whether there is a contaminant in a product and identify it. Using this expertise, it is possible to analyse a sample to determine what it contains and how much is in it. As an example, an unusual impurity was found in human growth hormone, which was determined to be a modification of the protein containing an additional sulphur atom in a disulphide bond. In most cases, a set of samples, which are implicated in an adverse event, are compared with normal samples. Any anomalous components are noted and identified, usually by tandem mass spectrometry (MS/MS). Then, an impurity is isolated and the identity confirmed by nuclear magnetic resonance (NMR) spectroscopy. The combination of MS/MS and NMR provides an extremely powerful approach to solving structure and identification problems.

The Proteomics and Mass Spectrometric Analysis laboratory is working to:

  • Identify potential contaminants in lots (early on or in response to reports of adverse reactions) to help identify, prevent and/or recall contaminated or unsafe products;
  • Develop new detection techniques to profile biologics, which can also help to update and improve national and international standards for active ingredients and products;
  • Deliver rapid and accurate analyses, which are crucial for providing information that helps others understand health issues. We help protect populations against unsafe products and reduce the occurrence of adverse drug reactions.

 

Research highlight 2: Disease diagnosis by proteomics - Quebec platelet disorder

The Centre for Vaccine Evaluation (CVE) applies expertise in protein separation to the study of blood and blood products and biologics. For example, expertise in proteomic methods helped to diagnose a rare blood disorder - Quebec Platelet Disorder (QPD). The condition was first identified in a small population in Quebec. Medical genetics show that QPD is a rare genetic disorder that affects blood clotting. Since the condition is rare and not readily diagnosed, it can be challenging to identify, especially in cases outside the region where the disease was initially found. When a family of patients in British Columbia displayed unusual bleeding symptoms, CVE scientists collaborated with researchers from the Canadian Blood Services and used a proteomic analysis of the blood platelet proteins to reveal anomalies in concentrations of specific proteins. This information provided crucial insights that led to the identification of the disorder in those patients.

Work in this area suggests that:

  • Proteomic research aids in the diagnosis of certain conditions by narrowing the possibilities to a condition that correlates with a specific change in protein concentration;
  • Proteomic studies help us identify which proteins are associated with or cause a specific disease. In the case of rare diseases, the diagnosis may be very difficult. With extra information on protein distribution and expression, we can provide the necessary additional insights.

 

Research highlight 3: A new, fast, and effective method for testing flu vaccines using proteomics

Influenza is a respiratory infection caused by the influenza virus. Getting an annual influenza vaccination (or flu shot) can help prevent an infection or reduce the severity of the illness.

Annual flu vaccines are composed of a mixture of proteins from three strains of flu: proteins from two A strains and one B strain. Flu proteins are the key ingredients of the vaccine. To produce flu proteins, individual virus strains are grown in chicken eggs and are then purified by laboratory methods. As a result, residual egg protein may be present in vaccines.

The influenza virus mutates very rapidly, which is why it is necessary to prime our immune systems each flu season with that season's flu vaccine. Each year, the World Health Organization provides information on what strains should be used. The regulatory authorities then provide the initial strains to be used by the manufacturers for the production of annual flu vaccines. To make sure that the vaccines have the correct ingredients, an antibody-based test is used. This antibody requires up to six weeks to prepare. Scientists at the Centre for Vaccine Evaluation (CVE) have developed a new proteomics-based approach to test the vaccines. Mass spectrometry (MS) and protein analysis expertise is used to determine what strains are present in the vaccine. With MS and proteomics expertise, the influenza proteins in the mixture can be identified in less than three days.

Work in this area may provide:

  • Analysis of influenza vaccines that does not require the use of antibodies, which usually take six weeks to produce;
  • Increased ability to detect residual egg proteins that have the potential to cause allergic/adverse reactions in some individuals;
  • Rapid alternative test methods in case of a pandemic.

 

For information about the lead scientist of this laboratory, please visit their  Directory of Scientists and Professionals profile.

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